EXPERIMENTALPARASITOLOGY
74,431-140
(1992)
A Protective Monoclonal Antibody with Dual Specificity Plasmodium falciparum and Plasmodium berghei Circumsporozoite Proteins
for
BARBARA J. SINA,* CRAIG WRIGHT,? RIPLEY BALLOU,$ AND MICHAEL HOLLINGDALE* *Biomedical Research Institute, 12111 Parklawn Drive, Rockville, Maryland 20852, U.S.A.; fllnivax Corporation, 12280 Wilkins Avenue, Rockville, Maryland 20852, U.S.A.; and #Department of Immunology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, U.S.A. SINA,B. J., WRIGHT,C.,BALLOU,R., AND HOLLINGDALE, M. 1992.Aprotective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Experimental Parasitology 74, 431-440. An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial iIIfCCtiOIL INDEX berghei;
0 1992 Academic Press, Inc. DESCRIPTORS AND ABBREVIATIONS:
Plasmodium
falciparum;
Plasmodium
Malaria; Circumsporozoite protein; Monoclonal antibodies; Sporozoite immunity; CS, circumsporozoite (protein); IFA, immunofluorescence assay; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline.
Immunization with irradiated sporozoites of human (Clyde et al. 1973), simian (Gwadz et al. 1979), and rodent (Nussensweig et al. 1969) malaria species induces specific immunity which protects against subsequent sporozoite challenge. Although the mechanisms mediating in antisporozoite immunity are not thoroughly understood, several lines of evidence suggest that anti-sporozoite antibody made predominantly against the repeat region of the circumsporozoite (CS) protein (Zavala et al. 1983) plays a role in protection. In vitro, anti-CS protein-specific monoclonal and polyclonal antibodies block sporozoite invasion of hepatoma cells (Hollingdale et al. 1982, 1984) and hepatocytes (Mazier et al.
1985). Incubation in vitro of Plasmodium knowlesi (Cochrane et al. 1982), P. falciparum, and P. vivax (Nardin et al. 1982) sporozoites with CS protein repeat-specific monoclonal antibodies neutralizes their infectivity in vivo and passive transfer of a P. berghei CS protein repeat-specific monoclonal antibody was shown to protect mice against sporozoite challenge (Potocnjak et al. 1980). On the basis of these results, vaccine candidates derived from the P. falciparum immunodominant repeat region of CS protein were developed and tested in human volunteers with generally disappointing results despite eliciting high titers of antibody to the CS protein repeat (Herrington et al. 1987; Ballou et al. 1987). Failure of the various CS protein repeat vaccine candidates
431 0014-4894/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
432
SINA ETAL.
to protect was thought to be due to the lack (ANKA clone) by feeding on anesthetized infected sporozoites (NF54 strain) of stimulation of T-cell immunity, but sub- outbred mice. P. falciparum were obtained from A. stephensi mosquitoes (Dutch sequently, passive transfer of polyclonal strain) infected by membrane feeding on cultured paranti-sporozoite antibody and CS protein asitized human erythrocytes containing mature gamevaccine-generated antibody were found to tocytes (Ponnudurai et al. 1982). Infected mosquitoes be largely not protective (Egan et al. 1987). were used to immunize mice by bite, to challenge exTherefore, an alternative explanation for perimental mice with P. berghei infection, or to isolate salivary gland sporozoites for infection of cultured the lack of efficacy of induced polyclonal hepatoma cells and ELISA or immunoblot antigen antibodies compared to monoclonal antipreparation. Sporozoites were isolated from mosquito bodies might be that the vaccine sequences salivary glands dissected aseptically in cold RPM1 media containing 10% fetal calf serum by tituration in a elicited populations of ineffectual antibodglass homogenizer and counted in a hemocytometer. ies. A variety of antibody populations Monoclonal antibodies. Anesthetized BALB/c mice which displayed specific binding in IFA or were immunized four times with sporozoites during in various solid-phase assays could have re- biting by several P. falciparum-infected mosquitoes sulted from immunization but were func- every 14 days. Postimmunization sera were tested by ELBA (Wirtz et al. 1987) for high antibody titer to P. tionally incapable of neutralizing sporozofalciparum CS protein repeat using the recombinant ite infectivity for hepatocytes in vivo. vaccine candidate R32tet,, (Young et al. 1985), which This paper describes an IgM monoclonal was kindly provided by Dr. Mitch Gross (Smith Kline, antibody (Mab 36) which was generated by Beecham, Inc.). Two immunized mice were splenecP. falciparum sporozoite immunization that tomized and their combined splenocytes fused to reacts with the CS proteins of both P. fal- P3X63Ag8.635NP myeloma cells with polyethylene ciparum and P. berghei, presumably on the glycol (Kohler and Milstein 1975). Fused cells were selected in hypoxanthine-, aminopterin-, and thymibasis of a shared amino acid sequence dine-containing media and screened for antibody to P. within their repeat regions (Dame et al. falciparum CS protein repeat by ELISA and subse1984; Weber et al. 1987). Mab 36 displayed quently by immunoblots containing (PNAP), peptide a high biological activity, i.e., the ability to coupled to BSA. Positive colonies were cloned twice block both species of sporozoites from in- by limiting dilution. Ascites was produced in pristine primed BALB/c mice (Hoogenraad et al. 1983). Monovading hepatoma cells in vitro and to pro- clonal antibody subclasses were determined and the vide passively transferred protection specific monoclonal antibody concentration was quanagainst P. berghei challenge in vivo. Mab 36 titated by standard ELISA methods. Mab 36 was purified by ammonium sulfate precipitation followed by was shown to bind epitopes also recognized by other protective P. falciparum and P. gel filtration. The IgG mouse P. berghei CS protein repeatberghei anti-CS protein repeat monoclonal specific monoclonal antibody 3Dll (Yoshida et al. antibodies. Previously, cross-reactive 1980) and the P. falciparum CS protein repeat-specific monoclonals have been raised against P. monoclonal antibodies 3D6 and 2AlO (Nardin et a/. 1982) were generously provided by Drs. Cochrane and falciparum CS protein but have not been reported to show protection against P. Nussensweig (New York University). Immunoblotting. After dissection, sporozoites were berghei infection in a murine model (Wirtz pelleted in a microfuge for 5 min and resuspended in et al. 1987; Burkot et al. 1984; Ballou, per- SDS sample buffer at a concentration of 5000 sporosonal communication). These results sug- zoites per microliter for electrophoresis. Ten percent gest that anti-CS protein-specific IgM anti- polyacrylamide-SDS gels of sporozoite proteins were bodies such as Mab 36 may play a role in electroblotted onto nitrocellulose filters and incubated with monoclonal antibody, which was subsequently protection against malarial sporozoite in- detected using iodinated or horseradish peroxidasefection. conjugated anti-mouse Ig antibody according to stanMATERIALSAND Sporozoite
isolation.
dard procedures.
METHODS
Anopheles
toes (India strain) were infected
stephensi
mosqui-
with P. berghei
Passive transfer of Mab 36. Outbred mice were injected intravenously via the tail vein with either PBS, ascites containing an irrelevant IgM monoclonal anti-
PROTECTIVE
ANTIBODY
FOR
body (Mab 2-27) specific for Pseudomonas aeruginosa (Wright et al., unpublished), or various concentrations of Mab 36 ascites antibody. After 30 min, individual mice were challenged by biting with 10 P. bergheiinfected mosquitoes. Erythrocytic parasitemia was detected by daily microscopic examination of Giemsastained tail blood smears. ISI assay. In vitro inhibition of P. falciparum and P. berghei sporozoite invasion of hepatoma HepG2-A16 cells by various concentrations of Mab 36 was determined in the assay described by Hollingdale et al. (1982, 1984). Epitope mapping. The linear peptide epitopes of the P. falciparum and P. berghei CS protein repeat bound by the monoclonal antibodies Mab 36,3Dll, 3D6, and 2AlO were mapped by the method of Geysen et al. (1984, 1987). Briefly, monoclonal antibody was incubated with a 96pin support plate containing all possible overlapping hexapeptides of a CS protein repeat region synthesized by Cambridge Research Biochemicals. Bound antibody was detected using horseradish peroxidase-conjugated anti-mouse Ig antibody essentially as described above for ELISA. RESULTS
Characterization of Mab 36. Antibodysecreting hybridomas were prepared from P. falciparum sporozoite-immunized mice and screened for the ability to bind the fouramino-acid repeated sequence of the CS protein of P. falciparum by ELISA. Mab 36 was subclassed as an IgM antibody and its ELISA binding was confirmed by immunoblotting against the P. falciparum CS protein repeat peptide (NANP), coupled to BSA (data not shown). Immunoblots of total sporozoite proteins from various malaria species were used to examine the specificity of Mab 36 binding. Figure 1 shows that Mab 36 reacted with both P. falciparum and P. berghei CS proteins on immunoblots. There was no apparent binding to other non-CS P. falciparum or P. berghei sporozoite proteins, mosquito salivary gland protein contaminants, or P. gallinaceum, P. vivax, or P. yoelii sporozoite proteins (data not shown). Biological activity of Mab 36. The biological activity of Mab 36 was tested in several assays. As previously shown for other antiCS protein repeat-specific monoclonal an-
P.falciparum
AND
433
P. berghei Pb Pf
FIG. 1. Western blots of total sporozoite protein. Monoclonal antibodies Mab 36 and 2AlO were incubated with Western blots containing total P. berghei (p,,) and P. falciparum (pr) sporozoite protein. Bound antibody was detected using hydrogen peroxidaseconjugated anti-mouse antibody.
tibodies (Vanderberg et al. 1969; Cochrane et al. 1976), Mab 36 was shown to induce the CS precipitin reaction after incubation with live sporozoites (data not shown) and to bind to sporozoite-infected hepatoma cells as detected by immunoperoxidase staining (Fig. 2). Mab 36 was shown to block the invasion of hepatoma cells by P. falciparum and P. berghei sporozoites in vitro by 50% at concentrations of ~0.1 and 0.02 t&ml, respectively (Table I). The antiP. falciparum CS protein repeat-specific monoclonal antibody 2AlO and the anti-P. berghei CS protein repeat-specific monoclonal antibody 3Dll have been reported to show 50% inhibition of invasion at l-2 pg/ ml (Hollingdale et al. 1982, 1984). Therefore, Mab 36 displayed approximately loto loo-fold better inhibitory activity against live sporozoites . To further test biological activity, Mab 36 was administered intravenously to mice that were subsequently challenged by biting with P. berghei-infected mosquitoes. Table II shows that Mab 36 provided complete protection against P. berghei infection at doses of 160 pg. The minimum dosage required for protection was not determined but the lowest dose (16 pg) used displayed a lack of protection similar to that observed with the irrelevant IgM monoclonal antibody and PBS controls.
434
SINA ET AL. TABLE I Inhibition of Sporozoite Invasion Antibody
CS protein reactivity
Mab 2A10
P. falciparum
Mab 3Dll
P. berghei
Mab 36
P. falciparum P. berghei
Sporozoites P. P. P. P. P. P.
falciparum berghei falciparum berghei falciparum berghei
50% IS1 1-2 Kg/ml Negative Negative
2 rdml CO.1 &ml 0.02 &ml
Nore. Inhibition of sporozoite invasion (ISI) assays were performed by adding P. falciparum or P. berghei sporozoites to cultures of hepatoma cells in the presence of various concentrations of the monoclonal antibodies 36, 2A10, or 3Dl I. Sporozoite invasion was enumerated microscopically after immunoperoxidase staining. Percentage of invasion was calculated relative to invasion in control cultures that received no antibody. I& is the concentration of monoclonal antibody that reduced invasion to 50%.
FIG. 2. lmmunoperoxidase staining of exoerythrocytic stage P. berghei. Monoclonal antibody Mab 36 was incubated with confluent monolayers of hepatoma Hep G2-A16 cells fixed with methanol 48 hr after incubation with P. berghei sporozoites. Exoerythrocytic stage parasites which bound antibody were detected using hydrogen peroxidase-conjugated anti-mouse antibody and observed by light microscope examination (1000X magnification).
specificities. Mab 36 binds a subset of 5 linear epitopes of the P. berghei CS protein repeat sequence recognized by 3Dll (5 of 10 total 3D1 l-positive epitopes). It also recognizes 6 variant epitopes of the P. falciparum CS protein repeat, 3 of which overlap with the 7 peptides recognized by 2AlO. In contrast, the 6 P. falciparum epitopes recognized by Mab 36 represent a subset of the 10 epitopes recognized by 3D6. Therefore, all Mab 36 specificities are encomTABLE II Passive Transfer of Mab 36
Epitope specificity of Mab 36. Figure 3
presents the results of linear epitope mapping conducted with Mab 36 compared to the results obtained with other IgG CS protein repeat species-specific monoclonal antibodies. 3Dll was previously shown to protect mice from P. berghei sporozoite infection after passive transfer (Potocnjak et al. 1980) and 3D6 was previously shown to neutralize P. falciparum sporozoite infectivity to chimpanzees (Nardin et al. 1982). The binding of each monoclonal, quantitated as a spectrophotometric reading after ELISA, was graphed for each overlapping peptide hexamer variant of the CS protein repeat sequences for P. falciparum or P. antiberghei assayed. Each monoclonal body tested produced a unique pattern of
Antibody None Mab 2-67 Mab 36 None Mab 2-67 Mab 36
Dose ~I&
% Protected
P
value
Experiment 1 0 6.7 (l/15) 667 13.3 (2/15) 160 100 (H/15)
1.0000 0.0001
Experiment 2 0 27.7 (4/15) 400 46.7 (7/15) 480 100 (15115) 160 100 (15/15) 16 13.3 (2/15)
0.44860 0.00002 0.00002 0.64810
Note. In two separate experiments, PBS, Mab 36, or the IgM anti-k’. aeruginosa monoclonal antibody 2-27, which serves as a negative control, were injected intravenously into mice which were challenged 30 min later by the bite of 10 P. berg/z&infected mosquitoes. P values were derived by the x2 method.
PROTECTIVE ANTIBODY FOR P.falciparum
passed by those of the two monoclonal antibodies which have been shown to provide protection against P. falciparum and P. berghei sporozoite infection in vivo. When the sequences of all the peptides bound by Mab 36 are compared, a consensus epitope sequence of (NP)(NP)NXPP can be drawn. DISCUSSION
During studies of protection against P. berghei sporozoite infection by P. falciparum sporozoite immunization (Sina er al., submitted for publication) Mab 36 was generated by screening hybridomas for reaction with the repeat sequence from the P. falciparum CS protein. Immunoblotting showed that Mab 36 reacted with the CS proteins of both sporozoite species. Previously, in a comparative study of several P. falciparum CS protein monoclonal antibodies, some showed cross-reactivity with P. berghei sporozoites by IFA (Wirtz et al. 1987; Burkot et al. 1984). Mouse antisera made to synthetic peptides of the P. falciparum CS protein repeat coupled to BSA reacted with P. berghei sporozoites by IFA (Ballou et al. 1985) and rabbit sera generated to (NANP),-tetanus toxoid was reported to show cross-reaction with P. berghei CS protein (Zavala et al. 1985). Antibody cross-reactivity between the CS proteins of the two malarial species might be expected from comparison of their gene sequences, which shows that the immunodominant P. falciparum CS protein repeat (PNAN) (Dame et al. 1984) is included in the P. berghei eight-amino-acid CS protein repeat (DPAPPNAN) (Weber et al. 1987). Subsequent experiments with Mab 36 showed that it induces the CS precipitin reaction and inhibits P. falciparum and P. berghei sporozoite invasion of hepatoma cells by 50% in vitro at concentrations approximately lo- to loo-fold lower than those of previously reported IgG inhibitory monoclonal antibodies such as 2AlO and 3Dll (Hollingdale et al. 1982, 1984). Two previously reported P. berghei CS protein
AND P. berghei
435
repeat-specific IgM monoclonal antibodies were shown to be incapable of blocking invasion (Leland et al. 1984) whereas all hyperendemic area patient sera with more than 75% IS1 activity had IgM and/or IgG titers of more than l/800 to R32tet,, (Hoffman et al. 1986). Theoretically, IgM molecules, with 10 potential binding sites, could show as much as a 107-fold increase in binding energy when binding an antigen in a multivalent manner. In addition, the multivalent nature of the highly repeated CS protein immunodominant epitope may contribute to the increased inhibition of invasion observed with Mab 36. Bound pentameric Mab 36 could also block invasion more effectively by steric hindrance or masking the CS Nl region (Aley et al. 1986) or other sporozoite surface proteins involved in hepatocyte receptor recognition and entry mechanisms. Mab 36 provides complete protection against P. berghei infection by infected mosquito bite when passively transferred to outbred mice at 160~p,g doses but not at 16pg doses. Potocnjak et al. (1980) reported that lo-kg doses of monoclonal antibody 3Dll protected A/J mice against an intravenous administration of 1000 P. berghei sporozoites but a minimum of 300 pg was required to protect against 10,000 sporozoites. Egan et al. (1987) reported that a minimum of 100 p,g of another P. berghei CS protein-specific IgG monoclonal antibody was required to protect C57B1/6 mice against 500 intravenously injected sporozoites. Other P. berghei sporozoite crossreactive P. falciparum CS protein monoclonal antibodies tested did not protect mice from P. berghei infection (R. Ballou, personal communication). The differences in the passive transfer protocols make direct comparison of these experiments difficult. Various mouse strains show large differences in susceptibility to intravenous P. berghei sporozoite infection (Jaffe et al. 1990). The route of sporozoite administration may also be critical. Recent reports in-
436
SINA ET AL. A 2.0 1.8 1.6 1.4 1.2 1 .o 0.8 0.6 0.4 0.2 0.0 ,-
2.0 1.8
?-
9 P
T-
1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2
FIG. 3. Linear epitope mapping. (A) Monoclonal antibody Mab 36 (black bars), monoclonal antibody 2AlO (white bars); (B) monoclonal antibody Mab 36 (black bars), monoclonal antibody 3D6 (white bars); (C) monoclonal antibody Mab 36 (black bars), monoclonal antibody 3Dll (white bars). The X-axis lists the hexameric peptide variants of the P. jiilciparum (A and B) or P. berghei (C) CS protein repeat sequence which were tested by ELISA from which the relative absorbance at 580 nm is plotted on the Y-axis. D represents a summary of the number of epitope variants shared between the monoclonal antibodies tested. (*) A human IgM monoclonal antibody derived from a volunteer protected against malaria by the CS repeat vaccine R32tet,, (Sadoff ef al., unpublished results).
dicate that on average very few sporozoites are inoculated during the bite of an infected mosquito (Rosenberg et al. 1990; Ponnudurai et al. 1989) and as few as five infected mosquito bites will guarantee 100% infec-
tion in outbred mice (Hollingdale, unpublished) or humans (Rickman et al. 1990). Several experimental observations suggest that sporozoites are not directly injected into the host bloodstream by mosquitoes
PROTECTIVE
ANTIBODY
P.fulciparum
FOR
AND
P. berghei
437
1.64 1.4 1.2
1 .o 0.8 0.6 0.4 0.2 0.0
P.falciparum
P.berghei
CS protein
CS protein
repeat
repeat
epitope
epitope
variants
variants
FIG. 3-Continued
but may instead be carried to the liver via the lymphatic system (Ponnudurai ef al. 1989; Rossignol el al. 1984; Boyd 1949). After intravenous injection of sporozoites, approximately 90% were found sequestered in the mouse spleen (Ferreira et al. 1986). In light of these recent findings, the efficacy of antibody based protection against sporozoite infection should be re-evaluated using sporozoite infection by mosquito bite. CS proteins contain an immunodominant region containing repeated epitopes (Zavala et al. 1983). Inhibition of sporozoite de-
rived (Zavala et al. 1985) or recombinant CS protein (Dame et al. 1985) binding of monoclonal antibodies by synthetic peptides of increasing multimers of the repeat sequence NANP showed that the epitope consists of two to three repeats. Since most linear B-cell epitopes have been found to consist of five to eight amino acids, the specific epitope sequence bound by antibody can be determined by an ELISA method employing a template containing an overlapping series of peptide variants which are synthesized simultaneously on template
438
SINA ET AL.
pins designed to fit a standard 96-well microtiter plate (Geysen et al. 1984, 1987). The results presented in Fig. 3 using this method indicate that a unique pattern of variant epitopes can be recognized by various monoclonal antibodies generated against the same CS protein repeat, as also recently reported by Burkot et al. (1991) with six P. falciparum CS protein-specific monoclonal antibodies. The monoclonal antibody 2A10 recognized the same set of hexameric peptide sequences in both studies, sharing three epitope specificities with Mab 36. As expected from the observed cross-reactivity, Mab 36 binds a hybrid subset of the epitopes recognized by the P. berghei and P. falciparum CS-specific monoclonal antibodies. Interestingly, the epitope specificities of Mab 36, which was shown to be protective after passive transfer, are completely encompassed by the monoclonal antibodies shown to be associated with protection in vivo; i.e., the DGIGM human monoclonal antibody (Sadoff, unpublished results), which was isolated from a protected individual from a CS repeat vaccine trial (Ballou et al. 1987), monoclonal antibody 3D6, which was shown to neutralize the infectivity of P. fulciparum sporozoite in chimpanzees (Nardin et al. 1982), and monoclonal antibody 3Dll protected mice from infection after passive transfer (Potocnjak et al. 1980). In comparison, it has been shown that passive transfer of polyclonal anti-sporozoite antibody or anti-CS vaccine antibody did not fully protect mice against P. berghei sporozoite challenge (Spitalny et al. 1976; Egan et al. 1987). In a recent study (Charoenvit et al. 1991), saimiri monkeys immunized with recombinant P. vivax CS vaccine were not protected despite developing high antivaccine antibody titers, possibly in part because this antibody did not recognize the CS repeat epitope specificity of a monoclonal antibody shown to be protective in the same animal model. The epitope specificities of protective anti-CS protein repeat
monoclonal antibodies may offer a standard to be compared to the antibody populations elicited by vaccine candidates. The mechanisms involved in protective preerythrocytic stage immunity are not thoroughly understood but the isolation of a protective anti-CS repeat-specific IgM monoclonal lends credence to the theory that a T-cell-independent B-cell response to the CS antigen could play a role (Scofield 1991). ACKNOWLEDGMENTS The authors are grateful for the dedicated technical assistance provided by Angela Appiah, Leslie Graves, David Hayes, Bea Akintilo, and Olga Muratova. This work was supported by U.S.A.I.D. Contract DPE0453-C-00-4027-00.
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Transactions of the Royal Society of Tropical Medicine and Hygiene 78, 639-640. MAZIER, D., BEAUDOIN, R. L., MELLOUK, S., DHUILHE, P., TEXIER, B., TROSPER, J., MIHGEN, F., LANDAU, I., PAUL, C., BRANDICOURT, O., GUGUEN-GUILLOUZO, C., AND LANGLOIS, P. 1985. Complete development of hepatic stages of Plasmodium falciparum in vitro. Science 227, 44U2. NARDIN, E. H., NUSSENSWEIG, V., NUSSENSWEIG, R. S., COLLINS, W. E., HARINASUTA, K. T., TAPCHAISRI, P., AND CHOMCHARN, Y. 1982. Cir-
cumsporozoite
proteins of human malaria parasites
SINA ET AL.
440
Plasmodium falciparum and Plasmodium vivax. Journal of Experimental Medicine 156, 20-30. NUSSENSWEIG, R. S., VANDERBERG, J. P., Mom, H.,
C. 1969. Specificity of protective improduced by X-irradiated Plasmodium berghei sporozoites. Nature 222, 488-489. PONNUDUIZAI, T., MEUWISSEN, T. H. E. TH., LEEUWENBERG, A., VERHAVE, J. P., AND LENSEN, A. H. W. 1982. The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolated infective to mosquitoes. AND
ORTON,
munity
Transactions of the Royal Society icine and Hygiene 76, 242. PONNUDURAI,
T.,
LENSEN,
G. J. A., BOLMER, T. H. E. TH. 1989.
A. M.
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W., AND
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POTOCNJAK, P., YOSHIDA, AND NUSSENSWEIG, V.
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GEMERT,
MEUWISSEN,
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and Hy-
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R. S.,
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P. A., RIBERIO, J. M. C., AND SPIELA. 1984. Increased intradermal probing time in sporozoite infected mosquitoes. American Journal of Tropical Medicine and Hygiene 33, 17-20. SCOFIELD, L. 1991. On the function of peptitive domains in protein antigens of Plasmodium and other eukaryotic parasites. Parasitology Today 7,99-105. SPITALNY, G. L., RIVERA-ORTIZ, C., AND NUSSENSWEIG, R. S. 1976. Plasmodium berghei: The spleen MAN,
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63, 295-300.
F.,
ZAVALA,
BELL, G. H., BURKOT, SER, K. M., BEAUDOIN,
CHAROENVIT,
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CAMPI., EsR. G.
1987. Comparative testing of monoclonal antibodies against Plasmodium falciparum sporozoites for ELISA development. Bulletin of the World Health Organization
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1980. Monovalent fragments (Fab) of monoclonal antibodies to sporozoite surface antigen (Pb 44) protect mice against malarial infection. Journal of Experimental Medicine 151, 1504-1513. RICKMAN, L. S., JONES, R. T., LONG, G. W., PAPARELLO, S., SCHNEIDER, I., PAUL, C. F., BEAUDOIN, R. L., AND HOFFMAN, S. L. 1990. Plasmodium falciparum infected Anopheles stephensi inconsistantly transmit malaria to humans. American Journal of Tropical Medicine and Hygiene 43, 441445. ROSENBERG, R., WIRTZ, R. A., SCHNEIDER, I., AND BURGE, R. 1990. An estimation of the number of malaria sporozoites ejected by a feeding mosquito.
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Sporozoite load of mosquitoes infected with Plasmodium falciparum. Transactions of the Royal Society giene 83, 67.
in sporozoite induced immunity to mouse malaria. Parasitology 40, 179-188. VANDERBERG, J., NUSSENSWEIG, R., AND MOST, H. 1969. Protective immunity produced by the injection of x-irradiated sporozoites of P. berghei. V. In vitro effects of immune serum on sporozoites. Military Medicine Supplement 134, 1183-l 190. WEBER, J. L., EGAN, J. E., LYON, J. A., WIRTZ, R. A., CHAROENVIT, Y., MALOY, W. L., AND HOCKMEYER, W. T. 1987. Plasmodium berghei: Cloning of the circumsporozoite protein gene. ExExperimental
YOSHIDA,
N.,
65, 39-45. NUSSENSWEIG,
R.
S.,
POTOCNJAK,
P.,
M. 1980. Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science 207, 71-73. NUSSENSWEIG,
YOUNG,
J. F.,
V.,
AND
AIKAWA,
HOCKMEYER,
LOU, W. R., WIRTZ, R. A., DOIN, R. L., HOLLINGDALE, L. H., DIGGS, C. L., AND
W.
T., GROSS,
TROSPER,
M.
M.,
BAL-
J. H., BEAUR., MILLER,
M. 1985. Expression of Plasmodium falciparum circumsporozoite protein in Escherichia coli for potential use in human malaria vaccine. Science 228,958-962. ZAVALA, F., COCHRANE, A. H., NARDIN, E. H., NIJSSENSWEIG, R. S., AND NUSSENSWEIG, V. 1983. Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. Journal of Experimental Medicine 157, 1947-1957. ZAVALA, F., TAM, J. P., COCHRANE, A. H., QUAKYI, I.,
NUSSENSWEIG,
R. S.,
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NUSSENSWEIG,
V.
1985. Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria. Science 228, 1436-1440. Received 20 September 1991; accepted with revision 27 February 1992
74,431-140
(1992)
A Protective Monoclonal Antibody with Dual Specificity Plasmodium falciparum and Plasmodium berghei Circumsporozoite Proteins
for
BARBARA J. SINA,* CRAIG WRIGHT,? RIPLEY BALLOU,$ AND MICHAEL HOLLINGDALE* *Biomedical Research Institute, 12111 Parklawn Drive, Rockville, Maryland 20852, U.S.A.; fllnivax Corporation, 12280 Wilkins Avenue, Rockville, Maryland 20852, U.S.A.; and #Department of Immunology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, U.S.A. SINA,B. J., WRIGHT,C.,BALLOU,R., AND HOLLINGDALE, M. 1992.Aprotective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Experimental Parasitology 74, 431-440. An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial iIIfCCtiOIL INDEX berghei;
0 1992 Academic Press, Inc. DESCRIPTORS AND ABBREVIATIONS:
Plasmodium
falciparum;
Plasmodium
Malaria; Circumsporozoite protein; Monoclonal antibodies; Sporozoite immunity; CS, circumsporozoite (protein); IFA, immunofluorescence assay; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline.
Immunization with irradiated sporozoites of human (Clyde et al. 1973), simian (Gwadz et al. 1979), and rodent (Nussensweig et al. 1969) malaria species induces specific immunity which protects against subsequent sporozoite challenge. Although the mechanisms mediating in antisporozoite immunity are not thoroughly understood, several lines of evidence suggest that anti-sporozoite antibody made predominantly against the repeat region of the circumsporozoite (CS) protein (Zavala et al. 1983) plays a role in protection. In vitro, anti-CS protein-specific monoclonal and polyclonal antibodies block sporozoite invasion of hepatoma cells (Hollingdale et al. 1982, 1984) and hepatocytes (Mazier et al.
1985). Incubation in vitro of Plasmodium knowlesi (Cochrane et al. 1982), P. falciparum, and P. vivax (Nardin et al. 1982) sporozoites with CS protein repeat-specific monoclonal antibodies neutralizes their infectivity in vivo and passive transfer of a P. berghei CS protein repeat-specific monoclonal antibody was shown to protect mice against sporozoite challenge (Potocnjak et al. 1980). On the basis of these results, vaccine candidates derived from the P. falciparum immunodominant repeat region of CS protein were developed and tested in human volunteers with generally disappointing results despite eliciting high titers of antibody to the CS protein repeat (Herrington et al. 1987; Ballou et al. 1987). Failure of the various CS protein repeat vaccine candidates
431 0014-4894/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
432
SINA ETAL.
to protect was thought to be due to the lack (ANKA clone) by feeding on anesthetized infected sporozoites (NF54 strain) of stimulation of T-cell immunity, but sub- outbred mice. P. falciparum were obtained from A. stephensi mosquitoes (Dutch sequently, passive transfer of polyclonal strain) infected by membrane feeding on cultured paranti-sporozoite antibody and CS protein asitized human erythrocytes containing mature gamevaccine-generated antibody were found to tocytes (Ponnudurai et al. 1982). Infected mosquitoes be largely not protective (Egan et al. 1987). were used to immunize mice by bite, to challenge exTherefore, an alternative explanation for perimental mice with P. berghei infection, or to isolate salivary gland sporozoites for infection of cultured the lack of efficacy of induced polyclonal hepatoma cells and ELISA or immunoblot antigen antibodies compared to monoclonal antipreparation. Sporozoites were isolated from mosquito bodies might be that the vaccine sequences salivary glands dissected aseptically in cold RPM1 media containing 10% fetal calf serum by tituration in a elicited populations of ineffectual antibodglass homogenizer and counted in a hemocytometer. ies. A variety of antibody populations Monoclonal antibodies. Anesthetized BALB/c mice which displayed specific binding in IFA or were immunized four times with sporozoites during in various solid-phase assays could have re- biting by several P. falciparum-infected mosquitoes sulted from immunization but were func- every 14 days. Postimmunization sera were tested by ELBA (Wirtz et al. 1987) for high antibody titer to P. tionally incapable of neutralizing sporozofalciparum CS protein repeat using the recombinant ite infectivity for hepatocytes in vivo. vaccine candidate R32tet,, (Young et al. 1985), which This paper describes an IgM monoclonal was kindly provided by Dr. Mitch Gross (Smith Kline, antibody (Mab 36) which was generated by Beecham, Inc.). Two immunized mice were splenecP. falciparum sporozoite immunization that tomized and their combined splenocytes fused to reacts with the CS proteins of both P. fal- P3X63Ag8.635NP myeloma cells with polyethylene ciparum and P. berghei, presumably on the glycol (Kohler and Milstein 1975). Fused cells were selected in hypoxanthine-, aminopterin-, and thymibasis of a shared amino acid sequence dine-containing media and screened for antibody to P. within their repeat regions (Dame et al. falciparum CS protein repeat by ELISA and subse1984; Weber et al. 1987). Mab 36 displayed quently by immunoblots containing (PNAP), peptide a high biological activity, i.e., the ability to coupled to BSA. Positive colonies were cloned twice block both species of sporozoites from in- by limiting dilution. Ascites was produced in pristine primed BALB/c mice (Hoogenraad et al. 1983). Monovading hepatoma cells in vitro and to pro- clonal antibody subclasses were determined and the vide passively transferred protection specific monoclonal antibody concentration was quanagainst P. berghei challenge in vivo. Mab 36 titated by standard ELISA methods. Mab 36 was purified by ammonium sulfate precipitation followed by was shown to bind epitopes also recognized by other protective P. falciparum and P. gel filtration. The IgG mouse P. berghei CS protein repeatberghei anti-CS protein repeat monoclonal specific monoclonal antibody 3Dll (Yoshida et al. antibodies. Previously, cross-reactive 1980) and the P. falciparum CS protein repeat-specific monoclonals have been raised against P. monoclonal antibodies 3D6 and 2AlO (Nardin et a/. 1982) were generously provided by Drs. Cochrane and falciparum CS protein but have not been reported to show protection against P. Nussensweig (New York University). Immunoblotting. After dissection, sporozoites were berghei infection in a murine model (Wirtz pelleted in a microfuge for 5 min and resuspended in et al. 1987; Burkot et al. 1984; Ballou, per- SDS sample buffer at a concentration of 5000 sporosonal communication). These results sug- zoites per microliter for electrophoresis. Ten percent gest that anti-CS protein-specific IgM anti- polyacrylamide-SDS gels of sporozoite proteins were bodies such as Mab 36 may play a role in electroblotted onto nitrocellulose filters and incubated with monoclonal antibody, which was subsequently protection against malarial sporozoite in- detected using iodinated or horseradish peroxidasefection. conjugated anti-mouse Ig antibody according to stanMATERIALSAND Sporozoite
isolation.
dard procedures.
METHODS
Anopheles
toes (India strain) were infected
stephensi
mosqui-
with P. berghei
Passive transfer of Mab 36. Outbred mice were injected intravenously via the tail vein with either PBS, ascites containing an irrelevant IgM monoclonal anti-
PROTECTIVE
ANTIBODY
FOR
body (Mab 2-27) specific for Pseudomonas aeruginosa (Wright et al., unpublished), or various concentrations of Mab 36 ascites antibody. After 30 min, individual mice were challenged by biting with 10 P. bergheiinfected mosquitoes. Erythrocytic parasitemia was detected by daily microscopic examination of Giemsastained tail blood smears. ISI assay. In vitro inhibition of P. falciparum and P. berghei sporozoite invasion of hepatoma HepG2-A16 cells by various concentrations of Mab 36 was determined in the assay described by Hollingdale et al. (1982, 1984). Epitope mapping. The linear peptide epitopes of the P. falciparum and P. berghei CS protein repeat bound by the monoclonal antibodies Mab 36,3Dll, 3D6, and 2AlO were mapped by the method of Geysen et al. (1984, 1987). Briefly, monoclonal antibody was incubated with a 96pin support plate containing all possible overlapping hexapeptides of a CS protein repeat region synthesized by Cambridge Research Biochemicals. Bound antibody was detected using horseradish peroxidase-conjugated anti-mouse Ig antibody essentially as described above for ELISA. RESULTS
Characterization of Mab 36. Antibodysecreting hybridomas were prepared from P. falciparum sporozoite-immunized mice and screened for the ability to bind the fouramino-acid repeated sequence of the CS protein of P. falciparum by ELISA. Mab 36 was subclassed as an IgM antibody and its ELISA binding was confirmed by immunoblotting against the P. falciparum CS protein repeat peptide (NANP), coupled to BSA (data not shown). Immunoblots of total sporozoite proteins from various malaria species were used to examine the specificity of Mab 36 binding. Figure 1 shows that Mab 36 reacted with both P. falciparum and P. berghei CS proteins on immunoblots. There was no apparent binding to other non-CS P. falciparum or P. berghei sporozoite proteins, mosquito salivary gland protein contaminants, or P. gallinaceum, P. vivax, or P. yoelii sporozoite proteins (data not shown). Biological activity of Mab 36. The biological activity of Mab 36 was tested in several assays. As previously shown for other antiCS protein repeat-specific monoclonal an-
P.falciparum
AND
433
P. berghei Pb Pf
FIG. 1. Western blots of total sporozoite protein. Monoclonal antibodies Mab 36 and 2AlO were incubated with Western blots containing total P. berghei (p,,) and P. falciparum (pr) sporozoite protein. Bound antibody was detected using hydrogen peroxidaseconjugated anti-mouse antibody.
tibodies (Vanderberg et al. 1969; Cochrane et al. 1976), Mab 36 was shown to induce the CS precipitin reaction after incubation with live sporozoites (data not shown) and to bind to sporozoite-infected hepatoma cells as detected by immunoperoxidase staining (Fig. 2). Mab 36 was shown to block the invasion of hepatoma cells by P. falciparum and P. berghei sporozoites in vitro by 50% at concentrations of ~0.1 and 0.02 t&ml, respectively (Table I). The antiP. falciparum CS protein repeat-specific monoclonal antibody 2AlO and the anti-P. berghei CS protein repeat-specific monoclonal antibody 3Dll have been reported to show 50% inhibition of invasion at l-2 pg/ ml (Hollingdale et al. 1982, 1984). Therefore, Mab 36 displayed approximately loto loo-fold better inhibitory activity against live sporozoites . To further test biological activity, Mab 36 was administered intravenously to mice that were subsequently challenged by biting with P. berghei-infected mosquitoes. Table II shows that Mab 36 provided complete protection against P. berghei infection at doses of 160 pg. The minimum dosage required for protection was not determined but the lowest dose (16 pg) used displayed a lack of protection similar to that observed with the irrelevant IgM monoclonal antibody and PBS controls.
434
SINA ET AL. TABLE I Inhibition of Sporozoite Invasion Antibody
CS protein reactivity
Mab 2A10
P. falciparum
Mab 3Dll
P. berghei
Mab 36
P. falciparum P. berghei
Sporozoites P. P. P. P. P. P.
falciparum berghei falciparum berghei falciparum berghei
50% IS1 1-2 Kg/ml Negative Negative
2 rdml CO.1 &ml 0.02 &ml
Nore. Inhibition of sporozoite invasion (ISI) assays were performed by adding P. falciparum or P. berghei sporozoites to cultures of hepatoma cells in the presence of various concentrations of the monoclonal antibodies 36, 2A10, or 3Dl I. Sporozoite invasion was enumerated microscopically after immunoperoxidase staining. Percentage of invasion was calculated relative to invasion in control cultures that received no antibody. I& is the concentration of monoclonal antibody that reduced invasion to 50%.
FIG. 2. lmmunoperoxidase staining of exoerythrocytic stage P. berghei. Monoclonal antibody Mab 36 was incubated with confluent monolayers of hepatoma Hep G2-A16 cells fixed with methanol 48 hr after incubation with P. berghei sporozoites. Exoerythrocytic stage parasites which bound antibody were detected using hydrogen peroxidase-conjugated anti-mouse antibody and observed by light microscope examination (1000X magnification).
specificities. Mab 36 binds a subset of 5 linear epitopes of the P. berghei CS protein repeat sequence recognized by 3Dll (5 of 10 total 3D1 l-positive epitopes). It also recognizes 6 variant epitopes of the P. falciparum CS protein repeat, 3 of which overlap with the 7 peptides recognized by 2AlO. In contrast, the 6 P. falciparum epitopes recognized by Mab 36 represent a subset of the 10 epitopes recognized by 3D6. Therefore, all Mab 36 specificities are encomTABLE II Passive Transfer of Mab 36
Epitope specificity of Mab 36. Figure 3
presents the results of linear epitope mapping conducted with Mab 36 compared to the results obtained with other IgG CS protein repeat species-specific monoclonal antibodies. 3Dll was previously shown to protect mice from P. berghei sporozoite infection after passive transfer (Potocnjak et al. 1980) and 3D6 was previously shown to neutralize P. falciparum sporozoite infectivity to chimpanzees (Nardin et al. 1982). The binding of each monoclonal, quantitated as a spectrophotometric reading after ELISA, was graphed for each overlapping peptide hexamer variant of the CS protein repeat sequences for P. falciparum or P. antiberghei assayed. Each monoclonal body tested produced a unique pattern of
Antibody None Mab 2-67 Mab 36 None Mab 2-67 Mab 36
Dose ~I&
% Protected
P
value
Experiment 1 0 6.7 (l/15) 667 13.3 (2/15) 160 100 (H/15)
1.0000 0.0001
Experiment 2 0 27.7 (4/15) 400 46.7 (7/15) 480 100 (15115) 160 100 (15/15) 16 13.3 (2/15)
0.44860 0.00002 0.00002 0.64810
Note. In two separate experiments, PBS, Mab 36, or the IgM anti-k’. aeruginosa monoclonal antibody 2-27, which serves as a negative control, were injected intravenously into mice which were challenged 30 min later by the bite of 10 P. berg/z&infected mosquitoes. P values were derived by the x2 method.
PROTECTIVE ANTIBODY FOR P.falciparum
passed by those of the two monoclonal antibodies which have been shown to provide protection against P. falciparum and P. berghei sporozoite infection in vivo. When the sequences of all the peptides bound by Mab 36 are compared, a consensus epitope sequence of (NP)(NP)NXPP can be drawn. DISCUSSION
During studies of protection against P. berghei sporozoite infection by P. falciparum sporozoite immunization (Sina er al., submitted for publication) Mab 36 was generated by screening hybridomas for reaction with the repeat sequence from the P. falciparum CS protein. Immunoblotting showed that Mab 36 reacted with the CS proteins of both sporozoite species. Previously, in a comparative study of several P. falciparum CS protein monoclonal antibodies, some showed cross-reactivity with P. berghei sporozoites by IFA (Wirtz et al. 1987; Burkot et al. 1984). Mouse antisera made to synthetic peptides of the P. falciparum CS protein repeat coupled to BSA reacted with P. berghei sporozoites by IFA (Ballou et al. 1985) and rabbit sera generated to (NANP),-tetanus toxoid was reported to show cross-reaction with P. berghei CS protein (Zavala et al. 1985). Antibody cross-reactivity between the CS proteins of the two malarial species might be expected from comparison of their gene sequences, which shows that the immunodominant P. falciparum CS protein repeat (PNAN) (Dame et al. 1984) is included in the P. berghei eight-amino-acid CS protein repeat (DPAPPNAN) (Weber et al. 1987). Subsequent experiments with Mab 36 showed that it induces the CS precipitin reaction and inhibits P. falciparum and P. berghei sporozoite invasion of hepatoma cells by 50% in vitro at concentrations approximately lo- to loo-fold lower than those of previously reported IgG inhibitory monoclonal antibodies such as 2AlO and 3Dll (Hollingdale et al. 1982, 1984). Two previously reported P. berghei CS protein
AND P. berghei
435
repeat-specific IgM monoclonal antibodies were shown to be incapable of blocking invasion (Leland et al. 1984) whereas all hyperendemic area patient sera with more than 75% IS1 activity had IgM and/or IgG titers of more than l/800 to R32tet,, (Hoffman et al. 1986). Theoretically, IgM molecules, with 10 potential binding sites, could show as much as a 107-fold increase in binding energy when binding an antigen in a multivalent manner. In addition, the multivalent nature of the highly repeated CS protein immunodominant epitope may contribute to the increased inhibition of invasion observed with Mab 36. Bound pentameric Mab 36 could also block invasion more effectively by steric hindrance or masking the CS Nl region (Aley et al. 1986) or other sporozoite surface proteins involved in hepatocyte receptor recognition and entry mechanisms. Mab 36 provides complete protection against P. berghei infection by infected mosquito bite when passively transferred to outbred mice at 160~p,g doses but not at 16pg doses. Potocnjak et al. (1980) reported that lo-kg doses of monoclonal antibody 3Dll protected A/J mice against an intravenous administration of 1000 P. berghei sporozoites but a minimum of 300 pg was required to protect against 10,000 sporozoites. Egan et al. (1987) reported that a minimum of 100 p,g of another P. berghei CS protein-specific IgG monoclonal antibody was required to protect C57B1/6 mice against 500 intravenously injected sporozoites. Other P. berghei sporozoite crossreactive P. falciparum CS protein monoclonal antibodies tested did not protect mice from P. berghei infection (R. Ballou, personal communication). The differences in the passive transfer protocols make direct comparison of these experiments difficult. Various mouse strains show large differences in susceptibility to intravenous P. berghei sporozoite infection (Jaffe et al. 1990). The route of sporozoite administration may also be critical. Recent reports in-
436
SINA ET AL. A 2.0 1.8 1.6 1.4 1.2 1 .o 0.8 0.6 0.4 0.2 0.0 ,-
2.0 1.8
?-
9 P
T-
1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2
FIG. 3. Linear epitope mapping. (A) Monoclonal antibody Mab 36 (black bars), monoclonal antibody 2AlO (white bars); (B) monoclonal antibody Mab 36 (black bars), monoclonal antibody 3D6 (white bars); (C) monoclonal antibody Mab 36 (black bars), monoclonal antibody 3Dll (white bars). The X-axis lists the hexameric peptide variants of the P. jiilciparum (A and B) or P. berghei (C) CS protein repeat sequence which were tested by ELISA from which the relative absorbance at 580 nm is plotted on the Y-axis. D represents a summary of the number of epitope variants shared between the monoclonal antibodies tested. (*) A human IgM monoclonal antibody derived from a volunteer protected against malaria by the CS repeat vaccine R32tet,, (Sadoff ef al., unpublished results).
dicate that on average very few sporozoites are inoculated during the bite of an infected mosquito (Rosenberg et al. 1990; Ponnudurai et al. 1989) and as few as five infected mosquito bites will guarantee 100% infec-
tion in outbred mice (Hollingdale, unpublished) or humans (Rickman et al. 1990). Several experimental observations suggest that sporozoites are not directly injected into the host bloodstream by mosquitoes
PROTECTIVE
ANTIBODY
P.fulciparum
FOR
AND
P. berghei
437
1.64 1.4 1.2
1 .o 0.8 0.6 0.4 0.2 0.0
P.falciparum
P.berghei
CS protein
CS protein
repeat
repeat
epitope
epitope
variants
variants
FIG. 3-Continued
but may instead be carried to the liver via the lymphatic system (Ponnudurai ef al. 1989; Rossignol el al. 1984; Boyd 1949). After intravenous injection of sporozoites, approximately 90% were found sequestered in the mouse spleen (Ferreira et al. 1986). In light of these recent findings, the efficacy of antibody based protection against sporozoite infection should be re-evaluated using sporozoite infection by mosquito bite. CS proteins contain an immunodominant region containing repeated epitopes (Zavala et al. 1983). Inhibition of sporozoite de-
rived (Zavala et al. 1985) or recombinant CS protein (Dame et al. 1985) binding of monoclonal antibodies by synthetic peptides of increasing multimers of the repeat sequence NANP showed that the epitope consists of two to three repeats. Since most linear B-cell epitopes have been found to consist of five to eight amino acids, the specific epitope sequence bound by antibody can be determined by an ELISA method employing a template containing an overlapping series of peptide variants which are synthesized simultaneously on template
438
SINA ET AL.
pins designed to fit a standard 96-well microtiter plate (Geysen et al. 1984, 1987). The results presented in Fig. 3 using this method indicate that a unique pattern of variant epitopes can be recognized by various monoclonal antibodies generated against the same CS protein repeat, as also recently reported by Burkot et al. (1991) with six P. falciparum CS protein-specific monoclonal antibodies. The monoclonal antibody 2A10 recognized the same set of hexameric peptide sequences in both studies, sharing three epitope specificities with Mab 36. As expected from the observed cross-reactivity, Mab 36 binds a hybrid subset of the epitopes recognized by the P. berghei and P. falciparum CS-specific monoclonal antibodies. Interestingly, the epitope specificities of Mab 36, which was shown to be protective after passive transfer, are completely encompassed by the monoclonal antibodies shown to be associated with protection in vivo; i.e., the DGIGM human monoclonal antibody (Sadoff, unpublished results), which was isolated from a protected individual from a CS repeat vaccine trial (Ballou et al. 1987), monoclonal antibody 3D6, which was shown to neutralize the infectivity of P. fulciparum sporozoite in chimpanzees (Nardin et al. 1982), and monoclonal antibody 3Dll protected mice from infection after passive transfer (Potocnjak et al. 1980). In comparison, it has been shown that passive transfer of polyclonal anti-sporozoite antibody or anti-CS vaccine antibody did not fully protect mice against P. berghei sporozoite challenge (Spitalny et al. 1976; Egan et al. 1987). In a recent study (Charoenvit et al. 1991), saimiri monkeys immunized with recombinant P. vivax CS vaccine were not protected despite developing high antivaccine antibody titers, possibly in part because this antibody did not recognize the CS repeat epitope specificity of a monoclonal antibody shown to be protective in the same animal model. The epitope specificities of protective anti-CS protein repeat
monoclonal antibodies may offer a standard to be compared to the antibody populations elicited by vaccine candidates. The mechanisms involved in protective preerythrocytic stage immunity are not thoroughly understood but the isolation of a protective anti-CS repeat-specific IgM monoclonal lends credence to the theory that a T-cell-independent B-cell response to the CS antigen could play a role (Scofield 1991). ACKNOWLEDGMENTS The authors are grateful for the dedicated technical assistance provided by Angela Appiah, Leslie Graves, David Hayes, Bea Akintilo, and Olga Muratova. This work was supported by U.S.A.I.D. Contract DPE0453-C-00-4027-00.
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ALEY, S. B., HOLLINGDALE,
BATES,
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related to the intermediate host. In “Malariology,” Vol. 1, pp. 551-607. Saunders, Philadelphia. BURKOT, T. R., ZAVALA, F., GWADZ, R. W., COLLINS, F. H., NKJSSENSWEIG, R. S., AND ROBERTS,
D. R. 1984. Identification of malaria infected mosquitoes by a two site enzyme linked immunoabsorbant assay. American Journal of Tropical Medicine and Hygiene 33, 227-23 1. BURKOT, T. R., DA, Z. W., GEYSEN, H. M., WIRTZ, W. R., AND SAUL, A. 1991. Fine specificities of monoclonal antibodies against the Plasmodium falciparum circumsporozoite protein: Recognition of
PROTECTIVE
ANTIBODY
both repetitive and non-repetitive Immunology 13, 161-170.
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